Scott DyerThe Procter & Gamble CompanyChristianSchlechtriemand Christoph SchaefersFraunhofer IMEGordon CashUSEPA ContributorDifficult to Test Substances Workshop10 September 2014Mention of trade names, products, or services does not convey official EPAapproval, endorsement, or recommendation.ContentsHigh Production Volume (HPV) Program – OECDU.K. = Competent AuthorityDefining the class of materialsChainlengthsEnvironmental SourcesPhys/Chem Properties and EcotoxicityEffect of Alkyl Chain length: Water Sol. and EcotoxicityTrials and tribulationsSolving the program: Fraunhofer InstituteAliphatic Alcohol FamilyAliphatic alcohols category covering linear andessentially-linear monohydric alcohols with carbonchainlengths of C6 to C22.Essentially-linear alcohols are primarily mono-branched at the 2-position with the branching beingpredominantly methyl.Cn-C-C-C-C-C-OHCmn=1-16m is predominantly 0 or 1Sources of Alcohols in the EnvironmentAnionic andNonionicAlcohol-basedsurfactantsOther consumerproductsOther potential inputscontaining wax estersor fatty acidsFree fatty alcoholsWatercolumnproductionBenthicsedimentproductionIn situsynthesisWWTPTerrestrialrunoffPlant waxesFree fatty alcoholsWax estersFatty acidsIn situbacterialsynthesisFree fatty alcoholsmeasured in thewater column forrisk assessmentWater Solubility: SPARC vs. Measured n-OHs10100100010000

Solubility (mg/l)Estimate

Solubility (mg/L)0.000010.00010.0010.010.11106 8 10 12 14 16 18 20 22Carbon #

Solubility (mg/l)EstimateMeasured

Solubility (mg/L)Acute Toxicity: Effect of Water Solubility0.0010.010.11

Acute toxicity (mol/l)Fish (96 h LC50)Daphnia (48 h EC50)Daphnia (24 h)Other invert acuteAlgae (72/96 h EC50)Fish (96h LC50)Daphnia (48h EC50)Daphnia (24h EC50)Other Invert acute0.000000010.00000010.0000010.000010.00011 2 3 4 5 6 7 8logKow

Acute toxicity (mol/l)Algae (72/96 h EC50)Algae 48 hFish regressionInvertebrate regressionDaphnia regressionKonemannSolubility (SPARC)C13 (Kow 5.3)Estimated Chronic SARAlgae (72/96h EC50)Algae (48h EC50)Fish RegressionInvertebrate regressionDaphnia regressionKonemannSolubility (SPARC)Trials and TribulationsDevelopment of a chronic toxicity programDaphnia magnaTask 1generation of stable solutionsContractor #1: well known European contract laboratoryHighly experienced in highly hydrophobic chemicals(e.g., pesticides)Use of a flow-through generator columnResultsLack of ability to achieve stable concentrationsLarge variability and increased loss through timeEvidence of biodegradationTask 2seek another laboratory’s expert helpFraunhoferInitial Dialogue with FraunhoferHow to maintain concentrations of highly hydrophobic andquickly biodegrading substances?How to achieve test solutions as close to the water solubilitylimit as possible?Potential chronic effects ~ water solublity limits forchainlengths >C13Flow-through failed (why, issues, etc.)Microbial contamination in the test vesselsPotential Sources: Air, Water, Food, Test OrganismsHigh exchange rate would be necessary havingsignificant impact on pelagial crustaceans (e.g.,Daphnia)Performing Daphnia Reproduction Tests(OECD 211) with Long-Chain Fatty Alcohols© Fraunhofer IMEChristian Schlechtriem, Uwe Boshof, Heinrich Jürling, Christoph SchäfersD. MagnaReproduction Test OverviewThetest was performed in tightly closed100mL-vessels (single exposure accordingA saturated solution daily generated by stirringthe test substance in test media under slow stirconditions under sterile conditions, served ashighest concentration and was serially diluted toprepare the other concentrations.Thetest was performed in tightly closed100mL-vessels (single exposure accordingto OECD 211).Each daythe test organisms weretransferred to fresh media containing food.Analysis for the test concentrations:Saturated solution, all freshly prepared testsolutions and pooled aged solutions.Analysis of Test Solutions•All test concentrations were sampled for chemical analysis threetimes a week•A 500 mL aliquot of the fresh solutions•After 24 h, at the next renewal, the aged test liquids were pooled(vessels 1-5 and 6-10)•The analyte was extracted liquid-liquid partitioningwith n-hexane.•Samples were derivatized with MSTFA, duplicatemeasurementswere performed by GC-MS using deuterated n-decanol orndodecanol as internal standard.•The method was validated in Daphnia test medium intheappropriate concentration range according to guidelineSANCO/-825/00 and SANCO/3029/99 (specificity, linearity,accuracy, precision, identity and limit of quantification (LOQ)N-Methyl-N-(trimethylsilyl)trifluoroacetamide(MSTFA)Preparation of Test Solutions: WAF ApproachAdd test compound to WAF bottle (loading = mg/L)Dissolve test compound in solvent (e.g. ethanol)Place aliquot of solution (e.g. 1 mL) into sterilized, still warm(> 50°C) 2 L mixing bottle (wet bottle sidewall only)Slowly rotate horizontally to cover a max. area ofglass walls,continue rotation until solvent has evaporated under nitrogenAdd sterile water obtained by autoclaving or sterile filtrationNote: pHmay change byautoclavingClose bottle, then stir slowly for up to 24h at room temperatureAfter stirring the vessels will be left to settle for at least 2hSaturated aqueous phase were taken out of the drainportThe first fraction (0-100 mL) was discardedPrepare dilution series for lower test concentrationsSaturate the surfaces of the test flasks with eluant, then discardFill test flasks, add daphnia and feed algaeRelate toxicity to measured test concentrations (not loadings)Pilot Trial I (Dodecanol)Prior to the OECD 211 studies, several pre-studiesincluding a nonGLPDaphniareproduction pilot study with Dodecanol andPentadcanol were performed to demonstrate the feasibility of (partsof) the study plan.C12 Dodecanol: First Alcohol Studied in the Project•Initial trials of C12 solutions checked for acuteD. magnatoxicityand for the concentrations after differentloadingsoConcentrationsoConsistent preparation of test solutionsoHighest conentration ~ water solubilityoMaintenance of test concentrations over timeoScreen/range-find sensitivities ofDaphniareproduction•Rapid loss of Dodecanol over time•Provoked a discussion about its kinetics.Kinetics StudyPurpose: Determine potential factors responsible for loss ofDodecanolPilot Trial I (Dodecanol): Results, Next Steps•Factors Investigated•Adsorption to glassware•Source of biodegradation•Air•Algae•Daphnia02040608010012004812162024

dodecanol concentration (% of initial)Kinetic 1AlgaeAlgaeAlgae+DaphnidAlgae+Daphnid..Dodecanol Kinetics Study04812162024

dodecanol concentration (% oftime (h)0204060801001200 4 8 12 16 20 24totalwateronly6080100120Kinetic 2Algae autoklavAlgae autoklavAlgaeAlgaeDodecanol Kinetics Study: Sources of Loss020400 4 8 12 16 20 24AlgaeDaphnid onlyDaphnid onlyWAF sterileWAF sterileC12 Kinetics Study: Conclusions1. Sterile WAF = no loss over time2. For C12-OH, algae provided the primary source ofalcohol lossthrough time due to adsorption and/or biodegradationAutoclaving improved the situation; initial loss was stillobserved3. Next Steps⇒Daily renewal of test media⇒At each transfer rinsing the daphnids with sterilized dilutionwater⇒Decision to perform the C15-OH pre-study using autoclavedalgaePre-Study: PentadecanolObjectives:same as with C12oConcentrationsoConsistent preparation of test solutionsoHighest concentration ~ water solubilityoMaintenance of test concentrations over timeoScreen/range-find sensitivities ofDaphniareproductionoSpecial Notes:Lower water solubilitypotential for oil droplets,resulting in greater variability in concentration.Discussed used of filtration* to produceuniform/saturated solutionsCreated test solutions with and without filtrationArt: also investigated effect of technician on consistencyof sample preps* Apolar 0.2 μm filter (Millex FG, 50 mm, Millipore),which was wettedwith methanol prior to use to allow the passage ofthe aqueous solutionResults of Pre-Study with C15 (Pentadecanol)•Preparations withoutfiltration provided considerably higherconcentrations than model predictions.•This observation raised the question concerning therelationshipbetween the water soluble and non-water soluble test material(droplets) which were analytically detected in thetest medium butcould not be distinguished•Could the higher concentrations affect toxicity (acute vs. chronic)to Daphnia via physical effects (droplets)?Acute non-GLP toxicity tests were performed at higher loadingswithout filtration (above saturation limit)Results:•Higher loadings = higher the measured concentrations (i.e., nosaturation was observed)•No acute effects were observed (no droplet effect)Chronic Pre-Study with PentadecanolResults•All daphnids survived in the controls. However, thereproductiveoutput was very low (11 per female << 60 as given by OECD 211)•All daphnids seemed to be in bad condition, indicated by weakpigmentation and very small sizes. The time until the first brood wasvery high (13.3 d >> 9 d as given by OECD 211)Relationship between test substance losses andbacteria concentrations end of 24 h intervals•Test concentration01020304050607080901001 10 100 1000 10000 100000 10000001E+07bacteria/mL

losses (%)•Test concentrationcorrelated with oxygendepletion•Bacteria > 1000/mLnot found up to day 5•50-70% losses due toadsorption to glassware•No correlation between substance losses and bacteria density•High densities are always associated with high losses,•Losses below 60% only at low densities (but high losses can occur due to other reasons)Conclusions of Pre-Study with PentadecanolFeeding autoclaved algae toDaphniadid not meet survival andreproduction criteriaFinal considerations: live feed, renew test media dailyand minimizebacterial contamination by:A. Performing the entire test in a clean benchB. Ensuring an axenic feed algae cultureC. At each transfer rinse daphnids with sterilized dilution waterUsing sterile filtered artificial air for gentle aeration at the water surfaceto avoid adverse oxygen depletionDue to stressful conditions definitive tests shouldbe avoided in thewinter season (reduced reproductive output)Differences between C12-OH and C15-OH•C15-OH adsorbs much more to glass. New glassware reduces variability•C15-OH has to be tested in critical loadings abovewater solubility,potentially causing physical stressDefinitive Chronic StudiesResulting deviations of the test protocol fromOECD 2111. Minimizing degradation of test substance•Conduct tests under sterile conditions•Daily renewal of test solutions including feed algae•RinsingDaphniawith sterilized water at transfer2.Adapted guideline to cope with stress induced bydegradation of the test substance and counter-measures.•Gentle aeration with sterile-filtered air near the surfaceofthe test medium to avoid oxygen depletion•Modify OECD 211 criteria for 60 offspring/survivingfemale by 21 days to utilize historic OECD 202b criteria(60 neonates/control)•Extend study for up to 3 extra daysDay ofDaphniaStudy0 5 10 15 20

Measured Concentration (μg/L) Running Geometric Mean0.11101001000Day ofDaphniaStudy0 5 10 15 20

Measured Concentration (μg/L)0.1110100100025 μg/L (nominal)500 μg/L (nominal)DodecanolSchäfers C, et al. (2009)Environmental properties ofDefinitive Studies: Concentrations/TimeDay ofDaphniaStudyDay ofDaphniaStudy0 5 10 15 20

Measured Concentration (μg/L) Running Geometric Mean0.11101001000Day ofDaphniaStudyDay ofDaphniaStudy0 5 10 15 20

Measured Concentration (μg/L)0.1110100100024.4 μg/L (nominal)500 μg/L (nominal)TetradecanolEnvironmental properties oflong chain alcohols.Part 2: Structure-activityrelationship for chronic aquatictoxicity of long chain alcohols.Ecotoxicology andEnvironmental Safety, 72 (4):996–1005.Chronic toxicity of longchainalcoholsLevel 1 Level 2 Level 3 Level 4Control performanceC10 Nominal 152 411 1110 3000Mean offspring perMeasured initial % 80.2 80.9 86.7 93.3surviving femaleMeasured total % 15.3 25.3 33.1 40.9% effect on Survival 0 0 0 100% effect on Reproduction 0 8 1468.1 in 21 dC12 Nominal 25 69 185 500Measured initial % 80.8 80.7 88.2 106.8Measured total % 13.5 7.7 7.4 19.1% effect on Survival 0 0 0 60%effectonReproduction0810668.0 in 21 dReproduction68.0 in 21 dC14 Nominal 24 69 185 500Measured initial % 40.4 73.9 74.7 73.3Measured total % 6.4 5.2 6.9 15.3% reduction of survival 0 0 0 30% effect on Reproduction 3 8 15 1986.6 in 21 dC15 Nominal 30 65 139 300Measured initial % 50.4 86 75 80Measured total % 6.7 12.0 13.3 21.0% effect on Survival 0 0 0 0% effect on Reproduction 4.3 6.5 13.6 19.963.4 in 23 dSignificant effect on SurvivalSignificant effect on Reproduction >=10 %)Significant effect on Reproduction (< 10 %)No significant effectChronic Toxicity of Long Chain AlcoholsExposure to multiple measures (21),related to:Arithmetic meanmeasured initialconcentration10000

(μg/L)Water solubilityArithmetic mean measuredgeometric means of initial & agedconcentrations10000Water solubility10100100010 11 12 13 14 15 16

Concentration (μg/L)Chain length (#C)EC10EC50>NOEC immEC10Ecx...110100100010 11 12 13 14 15 16Dark Blue: ImmobilityPink: ReproductionConclusions•Cookbook methods for highly insolubles was not successful•Alcohols confirmed Narcosis/Non-Polar organic QSAR•The batch-renewals = best approach for highly bioegradables.•Minimize bacterial growth•Heroics (in view of the sponsors)Publications Telling the Full StorySeries of articles in Ecotoxicology and Environmental Safety, Vol 72 (2009)Reprints of the Schaefers et al, 2009 are availableat the WorkshopFraunhofer Institute for Molecular Biologyand Applied Ecology IME57392 Schmallenberg, Germanychristian.schlechtriem@ime.fraunhofer.dewww.ime.fraunhofer.deThank You